Translation rescue by targeting Ppp1r15a upstream open reading frame in vivo. Translation rescue by targeting Ppp1r15a upstream open reading frame in vivo

The eIF2 initiation complex is central to maintaining a functional translation machinery. Extreme stress such as life-threatening sepsis exposes vulnerabilities in this tightly regulated system, resulting in an imbalance between the opposing actions of kinases and phosphatases on the main regulatory subunit eIF2α. Here, we report that translation shutdown is a hallmark of established sepsis-induced kidney injury brought about by excessive eIF2α phosphorylation and sustained by blunted expression of the counterregulatory phosphatase subunit Ppp1r15a. We determined that the blunted Ppp1r15a expression persists because of the presence of an upstream open reading frame (uORF). Overcoming this barrier with genetic approaches enabled the derepression of Ppp1r15a, salvaged translation and improved kidney function in an endotoxemia model. We also found that the loss of this uORF has broad effects on the composition and phosphorylation status of the immunopeptidome that extended beyond the eIF2α axis. Collectively, our findings define the breath and potency of the highly conserved Ppp1r15a uORF and provide a paradigm for the design of uORF-based translation rheostat strategies. The ability to accurately control the dynamics of translation during sepsis will open new paths for the development of therapies at codon level precision. Overall design: Mice were subjected to a single-dose, 5 mg/kg LPS tail vein i.v. injection or vehicle saline and kidney tissues were harvested at indicated timepoints (0, 1, 4, or 16 hrs). Indicated cell lines with 60-80% confluency were harvested at baseline or 16 hrs after poly(I:C) transfection using lipofectamine 2000 (final working concentration 1.0 µg/mL).

eIF2起始复合物（eIF2 initiation complex）是维持功能性翻译系统的核心组分。诸如危及生命的脓毒症这类极端应激，会暴露这一严格调控系统的脆弱性，导致主调控亚基eIF2α上的激酶与磷酸酶的拮抗作用失衡。本研究发现，翻译停滞是已确立的脓毒症诱导肾损伤的标志性特征，其由过度的eIF2α磷酸化所引发，并因负调控磷酸酶亚基Ppp1r15a的表达受抑而持续存在。我们证实，Ppp1r15a的表达受抑会因上游开放阅读框（upstream open reading frame，uORF）的存在而持续存在。通过遗传学手段突破这一障碍，可解除Ppp1r15a的表达抑制，挽救翻译过程并改善内毒素血症模型小鼠的肾功能。我们还发现，该uORF的缺失会对免疫肽组的组成与磷酸化状态产生广泛影响，其影响范围远超eIF2α通路。综上，本研究明确了高度保守的Ppp1r15a uORF的作用范围与效能，并为基于uORF的翻译调节策略设计提供了范式。能够在脓毒症期间精准调控翻译动态，将为开发密码子级精准度的治疗手段开辟新路径。实验整体设计：小鼠接受单次5 mg/kg脂多糖（LPS）尾静脉注射，或给予生理盐水对照，在指定时间点（0、1、4或16小时）采集肾组织。将处于60%~80%汇合度的指定细胞系，在基线状态或使用脂质体转染试剂2000（终工作浓度1.0 μg/mL）转染聚肌胞苷酸（poly(I:C)）16小时后进行收集。